Abstract 453: Targeting <i>FOXL2C402G</i>vulnerabilities in adult type ovarian granulosa cell tumors
Authors: Thomas Welte, Veena Vuttaradhi, Eleonora Khlebus, Barrett Lawson, Robert T. Hillman, Nghi Nguyen, Mary Sobieski, Reid T. Powell, Clifford Stephan
Journal: Cancer Research (2023)

Abstract Background: Nearly all adult-type granulosa cell tumors of the ovary (aGCTs) carry the same oncogenic mutation affecting the second winged helix domain of the Forkhead family transcription factor FOXL2 (c.C402G; p.C134W). There are no currently available therapies that exploit the high prevalence of FOXL2 mutations in this disease. Objectives: Exploit FOXL2-C402G oncogenic addiction in order to identify FDA-approved compounds with differential potency in KGN-FOXL2WT/C402G cells and isogenic KGN-FOXL2—/— cells. Methods: CRISPR/Cas9 was used to generate isogenic derivatives of KGN, a cell line derived from a recurrent aGCT. Parental cells (KGN-FOXL2WT/C402G) or isogenic cells lacking both FOXL2 alleles (KGN-FOXL2—/—) were functionally characterized and loss of Foxl2 protein was confirmed by immunoblot. Two drug libraries comprising a total of 2,416 compounds (majority FDA approved) were screened for effectiveness in KGN-FOXL2WT/C402G cells and KGN-FOXL2—/— cells. Our assay included staining with DAPI, followed by automated image analysis of each well to enumerate nuclei/cell numbers. Based on Fraction Affected (FA) statistics, compounds were ranked according to effectiveness in KGN-FOXL2WT/C402G cells. FA-area under the curve (FA-AUC) cutoff &amp;gt;0.1 yielded 152 drug candidates, categorized into 13 drug classes. 40 drug candidates, including the top 1-4 hits in each drug class were chosen for further validation. Validation rate in the confirmatory screen was at 95%. A novel primary tumor organoid culture system for aGCT was developed and validated using whole exome sequencing and immunophenotype. Selected candidate compounds were further investigated in aGCT organoid cultures from two relapsed tumors. Results: We identified classes of drugs such as the inhibitors of Bromodomain family proteins and Hydroxy-Camptothecin analogs, with enhanced activity in KGN-FOXL2WT/C402G cells relative to KGN-FOXL2—/— cells. Unexpectedly glucocorticoids as a class were found to stimulate proliferation of KGN-FOXL2WT/C402G cells whereas they lacked this activity in KGN-FOXL2—/— cells. For 36 glucocorticoids present in the screen, FA-AUC ranged from -0.3191 to -0.1342 (highest proliferation index among all test compounds) in KGN-FOXL2WT/C402G cells compared to -0.0353 to +0.1667 in KGN-FOXL2—/— cells. In recurrent aGCT organoids, the high-potency glucocorticoid dexamethasone induced cell proliferation and co-treatment with glucocorticoids increased the IC50 of paclitaxel in KGN-FOXL2WT/C402G cells from 1.43 nM to 4.04 nM. Conclusion: Mutant FOXL2 mediates a proliferative response to glucocorticoids in cultured cells and primary aGCT organoids. Paclitaxel is a standard of care chemotherapy in the treatment of aGCTs and is often administered with glucocorticoid pre-medication. The adverse effect of glucocorticoids on paclitaxel efficiency shown here should thus be taken into consideration in the clinic. Citation Format: Thomas Welte, Veena Vuttaradhi, Eleonora Khlebus, Barrett Lawson, Robert T. Hillman, Nghi Nguyen, Mary Sobieski, Reid T. Powell, Clifford Stephan. Targeting FOXL2C402Gvulnerabilities in adult type ovarian granulosa cell tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 453.